{"database":"biostudies-literature","file_versions":[],"scores":null,"additional":{"submitter":["Zhang LS"],"funding":["National Heart, Lung, and Blood Institute","National Human Genome Research Institute"],"pagination":["3306-3312"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/S-EPMC9764283"],"repository":["biostudies-literature"],"omics_type":["Unknown"],"volume":["17(12)"],"pubmed_abstract":["Methods for the precise detection and quantification of RNA modifications are critical to uncover functional roles of diverse RNA modifications. The internal m<sup>7</sup>G modification in mammalian cytoplasmic tRNAs is known to affect tRNA function and impact embryonic stem cell self-renewal, tumorigenesis, cancer progression, and other cellular processes. Here, we introduce m<sup>7</sup>G-quant-seq, a quantitative method that accurately detects internal m<sup>7</sup>G sites in human cytoplasmic tRNAs at single-base resolution. The efficient chemical reduction and mild depurination can almost completely convert internal m<sup>7</sup>G sites into RNA abasic sites (AP sites). We demonstrate that RNA abasic sites induce a mixed variation pattern during reverse transcription, including G → A or C or T mutations as well as deletions. We calculated the total variation ratio to quantify the m<sup>7</sup>G modification fraction at each methylated site. The calibration curves of all relevant motif contexts allow us to more quantitatively determine the m<sup>7</sup>G methylation level. We detected internal m<sup>7</sup>G sites in 22 human cytoplasmic tRNAs from HeLa and HEK293T cells and successfully estimated the corresponding m<sup>7</sup>G methylation stoichiometry. m<sup>7</sup>G-quant-seq could be applied to monitor the tRNA m<sup>7</sup>G methylation level change in diverse biological processes."],"journal":["ACS chemical biology"],"pubmed_title":["m<sup>7</sup>G-quant-seq: Quantitative Detection of RNA Internal <i>N</i><sup>7</sup>-Methylguanosine."],"pmcid":["PMC9764283"],"funding_grant_id":["R01 HL155909","RM1 HG008935"],"pubmed_authors":["Ju CW","He C","Dai Q","Ye C","Chen L","Liu C","Wei J","Zhang LS"],"additional_accession":[]},"is_claimable":false,"name":"m<sup>7</sup>G-quant-seq: Quantitative Detection of RNA Internal <i>N</i><sup>7</sup>-Methylguanosine.","description":"Methods for the precise detection and quantification of RNA modifications are critical to uncover functional roles of diverse RNA modifications. The internal m<sup>7</sup>G modification in mammalian cytoplasmic tRNAs is known to affect tRNA function and impact embryonic stem cell self-renewal, tumorigenesis, cancer progression, and other cellular processes. Here, we introduce m<sup>7</sup>G-quant-seq, a quantitative method that accurately detects internal m<sup>7</sup>G sites in human cytoplasmic tRNAs at single-base resolution. The efficient chemical reduction and mild depurination can almost completely convert internal m<sup>7</sup>G sites into RNA abasic sites (AP sites). We demonstrate that RNA abasic sites induce a mixed variation pattern during reverse transcription, including G → A or C or T mutations as well as deletions. We calculated the total variation ratio to quantify the m<sup>7</sup>G modification fraction at each methylated site. The calibration curves of all relevant motif contexts allow us to more quantitatively determine the m<sup>7</sup>G methylation level. We detected internal m<sup>7</sup>G sites in 22 human cytoplasmic tRNAs from HeLa and HEK293T cells and successfully estimated the corresponding m<sup>7</sup>G methylation stoichiometry. m<sup>7</sup>G-quant-seq could be applied to monitor the tRNA m<sup>7</sup>G methylation level change in diverse biological processes.","dates":{"release":"2022-01-01T00:00:00Z","publication":"2022 Dec","modification":"2024-11-19T15:25:09.805Z","creation":"2024-11-19T15:25:09.805Z"},"accession":"S-EPMC9764283","cross_references":{"pubmed":["36398936"],"doi":["10.1021/acschembio.2c00792"]}}